Difference between revisions of "Part:BBa K3332034"

Revision as of 15:54, 27 October 2020

pLtetO-1

Repressed by tetR so that the downstream genes(LacI) can not be expressed.When ATc exists,the effect will disappear.We use it to express LacI in the presence of ATc.

Usage and Biology

pLtetO-1 promoter is a promoter from E. coli that can be repressed by the protein tetR(BBa_C0040 or BBa_K3332039).When there is anhydrotetracycline （ATc）, the repression will be inhibited.

pLtetO-1 promoter was used to express LacI(ssrAtag(mf-lon)) to construct a monostable kill switch. Without ATc, tetR is expressed to repress pLtetO-1, then E. coli will be killed.

Characterization:

We use two circuits to characterize pLtetO-1: pLtetO-1_E0420_pSB1C3 and J23106_P0140_ pLtetO-1_E0420_pSB1C3

The agarose gel electrophoresis images of target fragments are shown as below：

Fig 2. pLtetO-1_E0420_pSB1C3 and pUC57(BBa_K3332084) digested by EcoR I and Pst I (about 1026 bp)

Fig 3. J23106_P0140_ pLtetO-1_E0420_pSB1C3(BBa_K3332085) digested by SpeI and Pst I (about 3922 bp)

Note：E0420 is equal to B0034_E0020_B0015

Protocol:

1. Preparation of stock solution： dissolve ATc in absolute alcohol to make 1000× stock solution（the work concentration is 100ng/mL）

2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.

3.Add 4mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.

4.Add 200 µL ATc stock solution into the induction group when OD600 increased to 0.6.

5.Induce for 6 hours and the condition is the same as before.

6.Then, sampling 0.5mL culture in each tube. All samples are centrifuged at 12000rpm, 1 minute. Remove supernatant and add 500µL sterile PBS to resuspend.

7.Measure the fluorescence intensity（ECFP）and corresponding OD600 by 96-well plate reader, then calculate the fluorescence / OD value of each group.

Here is the result:

Fig.4 Fluorescence intensity/OD600 for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.

We discovered that 100ng/mL ATc can inhibit the repression of tetR on pLtetO-1, then turn on the expression of downstream gene of pLtetO-1.

Reference:

[1] Chan CT, Lee JW, Cameron DE, Bashor CJ, Collins JJ. 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat Chem Biol. 2016;12(2):82-86. doi:10.1038/nchembio.1979

Sequence and Features